Investigations within this project concern the cell biology of rare human genetic disorders and normal and abnormal intracellular processes. The research goal is to gain insight into changes in molecular function that underlie various genetic metabolic disorders and work towards treatments for these illnesses. The research focuses on four groups of rare disorders: 1. Disorders of sialic acid metabolism. The key enzyme in the sialic acid biosynthesis pathway is UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Dominant mutations in the allosteric site of GNE cause sialuria, characterized by overproduction of sialic acid. Recessive mutations in GNE cause the neuromuscular disorder hereditary inclusion body myopathy (HIBM). In the last year, we wrote an extensive review on all aspects of HIBM (Ref 3), performed a molecular modeling study on GNE mutations related to HIBM (Ref 5), wrote a HIBM case report (Ref 6), and performed biochemical analysis for the first gene therapy trial of a human HIBM patient (Ref 9). Our further studies focused on characterizing novel GNE isoforms in human and mice, subcellular localization and expression levels of GNE and other enzymes in the sialic acid pathway (GlcNAc 2-epimersase, CMP-sialic acid synthetase) and extensive characterization of the kidney and muscle phenotypes of our HIBM knock-in mouse model. In 2007, we characterized a knock-in HIBM mouse model and demonstrated that N-acetylmannosamine (ManNAc) rescues the phenotype of the homozygous mutant mice and is a promising treatment for human patients (J Clin Inv (2007) 117:1585-1594). Negotiations regarding an extensive toxicology study for an IND (investigational New Drug) application for the use of ManNAc are ongoing through the NIH-TRND (Therapies for Rare and Neglected Diseases) program, and our ManNAc patent is licensed to a ManNAc manufacturer, New Zealand Pharmaceuticals. This last year we tested alternative HIBM treatments on our murine model, including feeding with sialic acid pathway intermediates and GNE gene therapy, the results are being compiled for a manuscript. Our mouse model showed an unexpected kidney phenotype (of podocytopathy and glomerular membrane splitting) which was rescued by ManNAc feeding. We developed a lectin panel that characterized kidney the glycosylation status of our HIBM mouse model. We are now testing this panel on a variety of unexplained human renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane. These human disorders may benefit from ManNAc as a therapeutic agent. We plan to file an employee invention report for the developed lectin staining panel. 2. Disorders of 3-methylglutaconic aciduria (3MGA) presenting with or without optic atrophy. In 2001, our group isolated OPA3, a gene of unknown function responsible for Costeff syndrome, which is characterized by 3MGA and optic atrophy. We tested DNA from patients with/without 3MGA and/or isolated optic atrophy for mutations in OPA3. We investigated OPA3 function, and identified a novel OPA3 isoform with a rare dual mitochondrial and peroxisomal localization (Ref 7). We also extensively studied zebrafish models for Costeff syndrome to further elucidate OPA3 function of OPA3 (Ref 13). 3. Disorders of intracellular vesicle sorting and formation. These disorders include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, and other genetically unclassified disorders. Common clinical features are albinism due to defects in melanosomes and bleeding due to platelet defects. Our group investigates known and unknown HPS-causing genes, with the goal of better understanding the biology of the disease. Our group also catalogues the clinical and genetic characteristics of the distinct subtypes of HPS and related disorders. This last year we described the HPS-6 subtype (Ref 1), characterized two Chediak-Higashi syndrome (Refs 8,11) and one Griscelli syndrome patient (Ref 12), and described the importance of HPS subtyping in different populations (Refs 10, 14). To study the effects of HPS mutations, we perform cell biological studies on patients material (using immuno-fluorescence, immmuno-EM, and live cell imaging) to examine defective intracellular trafficking and sorting of proteins and organelles in HPS cells. Such cells fail to transport certain lysosomal proteins to their correct destinations, and HPS gene products are involved in recognizing the specific vesicles that give rise to lysosome-like organelles. 4. Genetic interstitial deletion syndromes. Routine mutation screening commonly involves PCR-based approaches followed by direct sequencing, as is a common application in our laboratory (Refs 2, 4, 15). Occurrence of larger genomic deletions may be missed by these approaches if the deletion breakpoints extend beyond the position of the PCR primers. In recessive disorders, this can lead to mistaking homozygosity for hemizygosity. Our group applied quantitative real-time PCR to detect hemizygosity and deletion breakpoints in a variety of rare disorders. - Hermansky-Pudlak syndrome: We identified patients with a large genomic deletion on the HPS1 locus (Griffin et al. Clin Genet (2005) 68, 23-30) and the HPS6 locus (Ref 1). - Holoprosencephaly: We tested patients by quantitative real-time PCR for submicroscopic deletions in candidate gene regions (Bendavid et al. J Med Genet (2006) 43, 496-500), supplementing multicolor FISH results. - Smith-Magenis syndrome (SMS): This disorder is mainly (greater than 95%) caused by an interstitial deletion of 17p11.2. Our group is currently performing quantitative real-time PCR to identify hemizygosity in key genes on 17p11.2 in 98 patients with SMS. Our results, in combination with FISH analysis and comparative genome hybridization (CGH)- arrays performed by collaborating groups, will shed light on the variable phenotype of SMS patients and genotype-phenotype correlations. In the future, these quantitative real-time PCR methods can be applied to other deletion syndromes (e.g., Jacobsen syndrome). In addition, we study the effects of haploinsufficiency on expression of the major SMS gene, retinoic acid induced (RAI1, manuscript submitted), and test if retinoic acid treatment affects RAI1 expression n patients cells. - FISH and qPCR analysis on 20 SMS patients identified no deletion in 17p11.2. Mutation analysis for single gene defects are ongoing (including RAI1 and RASD1) in our lab. We also performed CGH-arrays on these patients DNA to identify novel (micro) deletions or duplications. Preliminary results identified 4 novel chromosomal rearrangements (manuscript in preparation).